Weeds are major threats to the integrity of agricultural and natural environments due to their invasive and competing potential. Bioherbicides are substances based on natural compounds that are biodegradable and often have low residual effects. Plant species able to produce and release phytotoxic compounds may represent effective bioherbicide sources. Leaves of Quillaja lancifolia D.Don (formerly Q. brasiliensis (A.St.-Hil. & Tul.) Mart.) produce water-soluble specialized metabolites of the saponin class that could be evaluated for phytotoxic activity and potential as natural herbicides. This study was conducted to examine the impacts of Q. lancifolia total saponins aqueous extract (AE) at 4 and 10% (w/v) and of two combined reverse-phase chromatography purified saponin fractions (QB) at 1 and 2% (w/v) on morpho-physiological parameters of Lactuca sativa (lettuce) and Echinochloa crus-galli (barnyardgrass) in pre- and post-emergence bioassays. QB was only tested in pre-emergence assays. In pre-emergence bioassays, the germination rate and germination kinetics were determined. Post-emergence evaluations included effects on seedling morphology, root and shoot length, dry mass, and chlorophyll content. Osmotic potential and pH analyses ruled out roles for these factors in the observed responses. AE had a high inhibitory impact on the germination of both lettuce and barnyardgrass. QB at 1% and 2% (w/v) significantly decreased the growth of lettuce seedlings germinated in its presence by more than 10-fold. Phytotoxic effects on the post-emergence growth of lettuce, especially at the highest concentration tested of AE (10% w/v), was also observed. The presence of quillaic acid-based triterpene saponins in AE and QB was confirmed using different analytical methods. Therefore, both saponin-enriched fraction and aqueous extracts of Q. lancifolia inhibited tested plant growth and development. The water solubility of saponins and the availability of a sustainable source of these molecules from the leaves of cultivated young Q. lancifolia plants make them attractive candidates for use as bioherbicides.